Journal: The Journal of Biological Chemistry

Article Title: Transcription Factor NF-?B Regulates Expression of Pore-forming Ca 2+ Channel Unit, Orai1, and Its Activator, STIM1, to Control Ca 2+ Entry and Affect Cellular Functions *

doi: 10.1074/jbc.M111.275925

Figure Lengend Snippet: Effect of the NF-κB subunits p65, p50, and p52 on STIM1 and Orai1 transcript levels in HEK293 cells. Arithmetic means (±S.E., n = 14–17) of the abundance of mRNA encoding STIM1 (A) and Orai1 (B) in HEK293 cells expressing the NF-κB subunits p65/p50 or p65/p52. C, the success of NF-κB subunit transfection was determined utilizing RT-PCR and agarose gel electrophoresis. Upper left panel, shown is a representative agarose gel of cDNA after PCR extracted from HEK293 cells transfected with p65/p50 or p65/p52. TBP was used as an internal control. Upper right and lower panel, shown are the arithmetic means (±S.E., n = 14–17) of the mRNA abundance encoding p65 (upper right panel), p50 (lower left panel), or p52 (lower right panel) in HEK293 expressing p65/p50 or p65/p52 as analyzed by the 2−ΔΔCT method (27). * (p < 0.05) and ** (p < 0.01) indicate statistically significant differences (Student's unpaired t test or ANOVA).

Article Snippet: Transfection of Cells and NF-κB Inhibition HEK293 cells were transfected with the constitutively active mutant SGK1 S422D (hSGK1 SD in pIRES-EGFP), the inactive mutant SGK1 K127N (hSGK1 KN in pIRES-EGFP) ( 23 ), or the NF-κB subunits p65 ( 24 ), p50, and p52 (both obtained from imaGenes GmbH) using FuGENE HD transfection reagent (Roche Diagnostics) according to the manufacturer's instructions.

Techniques: Expressing, Transfection, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Control

Journal: The Journal of Biological Chemistry

Article Title: Transcription Factor NF-?B Regulates Expression of Pore-forming Ca 2+ Channel Unit, Orai1, and Its Activator, STIM1, to Control Ca 2+ Entry and Affect Cellular Functions *

doi: 10.1074/jbc.M111.275925

Figure Lengend Snippet: Effect of NF-κB on STIM1 and Orai1 protein abundance in HEK293 cells. A, confocal microscopy of STIM1 and Orai1 protein abundance in HEK293 cells is shown. Shown are HEK293 cells transfected with control plasmid (control), p65 and p50, or p65 and p52. The nuclei were stained with DRAQ5 (blue), Orai1 with a FITC-conjugated antibody (green, upper panel), and STIM1 with an Alexa Fluor® 546-conjugated antibody (green, lower panel). The experiment was completed three times. B, shown is a Western blot analysis of whole cell lysate in p65/p50- or p65/p52-transfected HEK293 cells showing STIM1 and Orai1 protein abundance. The blot was stripped and reprobed with tubulin to ensure equal loading and with p65, p50, and p52 to control transfection. Left, representative blot; middle, arithmetic means of STIM1/tubulin ratio (±S.E., n = 6); right, arithmetic means of Orai1/tubulin ratio (±S.E., n = 6). * (p < 0.05) and ** (p < 0.01) indicate statistically significant differences (Student's unpaired t test).

Article Snippet: Transfection of Cells and NF-κB Inhibition HEK293 cells were transfected with the constitutively active mutant SGK1 S422D (hSGK1 SD in pIRES-EGFP), the inactive mutant SGK1 K127N (hSGK1 KN in pIRES-EGFP) ( 23 ), or the NF-κB subunits p65 ( 24 ), p50, and p52 (both obtained from imaGenes GmbH) using FuGENE HD transfection reagent (Roche Diagnostics) according to the manufacturer's instructions.

Techniques: Quantitative Proteomics, Confocal Microscopy, Transfection, Control, Plasmid Preparation, Staining, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Transcription Factor NF-?B Regulates Expression of Pore-forming Ca 2+ Channel Unit, Orai1, and Its Activator, STIM1, to Control Ca 2+ Entry and Affect Cellular Functions *

doi: 10.1074/jbc.M111.275925

Figure Lengend Snippet: Effect of NF-κB subunit silencing on STIM1 and Orai1 transcript levels. A, arithmetic means (±S.E., n = 4–8) of the abundance of mRNA encoding STIM1 and Orai1 in HEK293 cells without and with silencing of p65, p50, and p52. * (p < 0.05), ** (p < 0.01), and *** (p < 0.001) indicate statistically significant differences (ANOVA). B, left panel, shown is a representative agarose gel of cDNA after RT-PCR extracted from HEK293 cells silenced with siRNA for p65, p50, or p52 to control the success of knock down. TBP was used as an internal control. Right panel, arithmetic means (±S.E., n = 3–8) of the mRNA abundance encoding p65 (black bar), p50 (light gray bar), or p52 (dark gray bar) in HEK293 cells compared with cells transfected with scramble siRNA (white bar) are shown. ** (p < 0.01) indicate statistically significant differences (ANOVA). C, Western blot analysis of whole cell lysate in HEK293 cells silenced with siRNA for p65, p50, or p52 is shown. As control, scrambled siRNA was utilized. The success of knockdown of p65, p50, or p52 was tested with specific antibodies. The blot was stripped and reprobed with tubulin to control equal loading. The experiment was completed five times. D, Western blot analysis of whole cell lysate in HEK293 cells silenced with siRNA for p65, p50, or p52 is shown. Upper left panel, shown is a representative blot of STIM1. Upper right panel, shown is a representative blot of Orai1. Lower left panel, shown is the arithmetic means of STIM1/tubulin ratio (±S.E., n = 5). Lower right panel, shown is the arithmetic means of Orai1/tubulin (±S.E., n = 5). ** (p < 0.01) indicate statistically significant differences (ANOVA).

Article Snippet: Transfection of Cells and NF-κB Inhibition HEK293 cells were transfected with the constitutively active mutant SGK1 S422D (hSGK1 SD in pIRES-EGFP), the inactive mutant SGK1 K127N (hSGK1 KN in pIRES-EGFP) ( 23 ), or the NF-κB subunits p65 ( 24 ), p50, and p52 (both obtained from imaGenes GmbH) using FuGENE HD transfection reagent (Roche Diagnostics) according to the manufacturer's instructions.

Techniques: Agarose Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction, Control, Knockdown, Transfection, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Transcription Factor NF-?B Regulates Expression of Pore-forming Ca 2+ Channel Unit, Orai1, and Its Activator, STIM1, to Control Ca 2+ Entry and Affect Cellular Functions *

doi: 10.1074/jbc.M111.275925

Figure Lengend Snippet: Effect of NF-κB on SOCE in HEK293 cells. A, shown are representative tracings of the fura-2 fluorescence ratio in fluorescence spectrometry during and after Ca2+ depletion and thapsigargin (1 μm) addition in HEK293 cells with or without treatment with Wogonin (100 μm). Arithmetic means (±S.E., n = 3, each experiment 14–21 cells) of peak (B) and slope (C) increase of the fura-2-fluorescence ratio in cells without (white bars) or with (black bars) treatment with Wogonin. D, shown are representative tracings of the fura-2 fluorescence ratio in fluorescence spectrometry during and after Ca2+ depletion and thapsigargin (1 μm) addition in HEK293 cells transfected with control plasmid (upper panel), p65/p50 (middle panel), or p65/p52 (lower panel). E, shown are the arithmetic means (±S.E., n = 3, each experiment 14–33 cells) of peak (Δ ratio) and slope (Δ ratio/s) of fura-2 fluorescence increase after readmission of Ca2+ in HEK293 cells transfected with control plasmid or p65/p50 with or without treatment with Wogonin (100 μm). F, shown are the arithmetic means (±S.E., n = 3, each experiment 14–33 cells) of peak (Δ ratio) and slope (Δ ratio/s) of fura-2 fluorescence increase after readmission of Ca2+ in HEK293 cells transfected with control plasmid or p65/p52 with or without treatment with Wogonin (100 μm). * (p < 0.05), ** (p < 0.001), *** (p < 0.001) indicate statistically significant differences (ANOVA).

Article Snippet: Transfection of Cells and NF-κB Inhibition HEK293 cells were transfected with the constitutively active mutant SGK1 S422D (hSGK1 SD in pIRES-EGFP), the inactive mutant SGK1 K127N (hSGK1 KN in pIRES-EGFP) ( 23 ), or the NF-κB subunits p65 ( 24 ), p50, and p52 (both obtained from imaGenes GmbH) using FuGENE HD transfection reagent (Roche Diagnostics) according to the manufacturer's instructions.

Techniques: Fluorescence, Transfection, Control, Plasmid Preparation

Journal: The Journal of Biological Chemistry

Article Title: Transcription Factor NF-?B Regulates Expression of Pore-forming Ca 2+ Channel Unit, Orai1, and Its Activator, STIM1, to Control Ca 2+ Entry and Affect Cellular Functions *

doi: 10.1074/jbc.M111.275925

Figure Lengend Snippet: Influence of NF-κB on the migration of HEK293 cells. Arithmetic means ± S.E. (n =) of the number of migrated HEK293 cells transfected with empty vector (control) or with NF-κB subunits p65/p50 or p65/p52 are shown. ** (p < 0.01) indicate significant differences (ANOVA).

Article Snippet: Transfection of Cells and NF-κB Inhibition HEK293 cells were transfected with the constitutively active mutant SGK1 S422D (hSGK1 SD in pIRES-EGFP), the inactive mutant SGK1 K127N (hSGK1 KN in pIRES-EGFP) ( 23 ), or the NF-κB subunits p65 ( 24 ), p50, and p52 (both obtained from imaGenes GmbH) using FuGENE HD transfection reagent (Roche Diagnostics) according to the manufacturer's instructions.

Techniques: Migration, Transfection, Plasmid Preparation, Control

Journal:

Article Title: Severe Acute Respiratory Syndrome Coronavirus Papain-Like Protease Ubiquitin-Like Domain and Catalytic Domain Regulate Antagonism of IRF3 and NF-?B Signaling ▿ †

doi: 10.1128/JVI.02220-08

Figure Lengend Snippet: PLP inhibits the NF-κB signaling pathway. (A) 293T cells were transfected with a 3xκB/luciferase reporter plasmid that reports NF-κB-mediated gene induction. The reporter was transfected with PLP, PLPΔUBL, or PLP-Sol-expressing plasmids (13). At 24 h after transfection, cells were treated wither either SeV (MOI of 5) (A), poly(I-C) (2 μg) (B), or TNF-α (10 ng) (C). Note that both PLP and PLP-Sol block NF-κB-mediated gene induction, while PLPΔUBL does not. (D) The effect of PLP on NF-κB signaling was assayed by Western blotting. 293T cells were transfected with GFP, PLP, PLPΔUBL, or UBL alone for 24 h. At 24 h posttransfection, cells were treated with 10 ng of TNF-α for 0, 15, 30, or 45 min and proteins were extracted at those time points. Western blots were analyzed for either total IKb or phospho-IKb. Below the blot is the numerical ratio of total IKb to phospho-IKb as quantified by IPLAB.

Article Snippet: IKKi, IKKβ, and TBK1 kinases were purchased from Invitrogen, while IκBα and NF-κB (p50) were purchased from Biomol International.

Techniques: Transfection, Luciferase, Plasmid Preparation, Expressing, Blocking Assay, Western Blot

Journal:

Article Title: Severe Acute Respiratory Syndrome Coronavirus Papain-Like Protease Ubiquitin-Like Domain and Catalytic Domain Regulate Antagonism of IRF3 and NF-?B Signaling ▿ †

doi: 10.1128/JVI.02220-08

Figure Lengend Snippet: Analysis of the effect of PLP on IκBα kinase activity and of PLP interactions with various NF-κB pathway proteins. (A) IκBα was incubated with IKKβ and ATP to induce phosphorylation and then analyzed by SDS-PAGE. The same reactions were carried out in the presence of PLP to determine if PLP interferes with kinase activity. Locations of nonphosphorylated and phosphorylated IκBα, PLP, and IKKβ are indicated by arrows. Molecular mass marker (M) sizes are shown to the right of the gel in kDa. (B) Native gel analysis of PLP interactions with NF-κB pathway proteins. Proteins were mixed, as indicated above the gels, and incubated in 50 mM HEPES (pH 7.5) for 10 min at 4°C and then diluted with 2× sample buffer before being loaded onto the gels. Locations of the individual proteins are indicated by arrows to the left and right of the gels.

Article Snippet: IKKi, IKKβ, and TBK1 kinases were purchased from Invitrogen, while IκBα and NF-κB (p50) were purchased from Biomol International.

Techniques: Activity Assay, Incubation, SDS Page, Marker

Journal:

Article Title: Severe Acute Respiratory Syndrome Coronavirus Papain-Like Protease Ubiquitin-Like Domain and Catalytic Domain Regulate Antagonism of IRF3 and NF-?B Signaling ▿ †

doi: 10.1128/JVI.02220-08

Figure Lengend Snippet: PLP from NL63 but not MHV is an IFN antagonist. PLPs from NL63 and MHV were assayed for their ability to inhibit IFN-β, NF-κB, and STAT1 signaling in 293T cells. (A) In this assay, 293T cells were transfected with N-RIG and either GFP-, MHV PLP1-, MHV PLP2-, or NL63 PLP2-expressing plasmids and an IFN-β/luciferase reporter plasmid. At 24 h posttransfection, cells were assayed for luciferase expression. The value of induction in GFP plus N-RIG was set at 100%, with all other values in relation to it. Notice that MHV PLP1 and -2 do not block IFN-β induction, while NL63 and SARS-CoV PLP do. (B) 293T cells were transfected with GFP-, MHV PLP1-, MHV PLP2-, or NL63 PLP2-expressing plasmids and an NF-κB/luciferase reporter plasmid. At 24 h posttransfection, cells were treated with 100 ng of TNF-α; 6 h later, cells were assayed for luciferase expression. The value of induction in GFP plus TNF-α was set at 100%, with all other values in relation to it. Notice that MHV PLP1 and -2 do not block IFN-β induction, while NL63 and SARS-CoV PLP do. (C) 293T cells were transfected with either GFP-, MHV PLP1-, MHV PLP2-, or NL63 PLP2-expressing plasmids and an ISRE/luciferase reporter plasmid. At 24 h posttransfection, cells were treated with 100 ng of IFN-β; 6 h later, cells were assayed for luciferase expression. The value of induction in GFP plus IFNβ was set at 100%, with all other values in relation to it. Notice that none of the expressed proteins inhibits ISRE induction.

Article Snippet: IKKi, IKKβ, and TBK1 kinases were purchased from Invitrogen, while IκBα and NF-κB (p50) were purchased from Biomol International.

Techniques: Transfection, Expressing, Luciferase, Plasmid Preparation, Blocking Assay

Journal:

Article Title: Severe Acute Respiratory Syndrome Coronavirus Papain-Like Protease Ubiquitin-Like Domain and Catalytic Domain Regulate Antagonism of IRF3 and NF-?B Signaling ▿ †

doi: 10.1128/JVI.02220-08

Figure Lengend Snippet: PLP catalytic mutants play a role in IFN antagonism. (A) 293T cells were transfected with an IFN-β/luciferase plasmid that reports IRF3-mediated gene induction. The reporter was cotransfected with N-RIG and GFP, PLP, or the PLP W1633A, C1651A, or D1826A mutants. At 24 h after transfection, cells were lysed and assayed for luciferase expression. (B) 293T cells were transfected with a 3xκB/luciferase plasmid that reports NF-κB-mediated gene induction. The reporter was cotransfected with GFP, PLP, or the PLP W1633A, C1651A, or D1826A mutants. At 24 h after transfection, cells were treated with 10 ng of TNF-α, and 6 h after treatment, the cells were lysed and assayed for luciferase expression. (C) PLP mutants were tested for their cleavage activity using the construct and experimental design described in the legend to Fig. ​Fig.8.8. The top panel is a Western blot for anti-GFP showing cleavage of the reporter protein, and the lower panel is a Western blot of the same extract probed with anti-HA antibody showing expression of the PLP mutants. (D) Deubiquitinase activities of the PLP mutants were assayed using the Flag-tagged ubiquitin assay described in the legend to Fig. ​Fig.8.8. The top panel is a Western blot probed with anti-Flag antibody showing Flag-ubiquitin conjugation to cellular proteins. WT, wild type. The bottom panel is a Western blot with anti-HA antibody showing expression of the PLP variants.

Article Snippet: IKKi, IKKβ, and TBK1 kinases were purchased from Invitrogen, while IκBα and NF-κB (p50) were purchased from Biomol International.

Techniques: Transfection, Luciferase, Plasmid Preparation, Expressing, Activity Assay, Construct, Western Blot, Ubiquitin Assay, Conjugation Assay

Journal:

Article Title: Rapid Protection of Gnotobiotic Pigs against Experimental Salmonellosis following Induction of Polymorphonuclear Leukocytes by Avirulent Salmonella enterica

doi: 10.1128/IAI.71.4.2182-2191.2003

Figure Lengend Snippet: Western blotting analysis of INT 407 whole-cell lysates probed with anti-NF-κB p50 antibody following incubation of monolayers with S. enterica serovar Typhimurium F98 Nalr (F98) or rough S. enterica serovar Infantis (S.inf.φr) at 37°C for 6 h (MOI = 10). +ve control, 1 μg of PMA/ml; −ve control, INT 407 cells incubated in RPMI 1640 medium without Salmonella or PMA; NFκB, p50 protein.

Article Snippet: The nitrocellulose was then incubated with rabbit anti-mouse NF-κB (p50) (1:200) (Autogenbioclear; Calne, Wilts, United Kingdom) for 60 min at room or ambient temperature on an end-to-end shaker.

Techniques: Western Blot, Incubation